It is known in the medical and biological arts that oxygen is a necessary component of animal metabolism. An oxygen supply is especially important in those bodily tissues undergoing continuous and critical activity, such as cardiac muscle, brain tissue and other nerve tissue. One example highlighting the desirability of having a method to determine oxygen concentration levels in animal tissues is diseases of the retina.
Retinal vascular disease is one of the leading causes of blindness in the United States and the major cause of vision loss throughout the world. Subtle changes in retinal tissue oxygenation, most notably tissue hypoxia, have been implicated as causal factors in the etiology of neovascularization in diabetic retinopathy, neovascularization following branch vein occlusion, sickle-cell anemia retinopathy, retrolental fibroplasia (retinopathy of prematurity), and hypertensive changes in the retinal vasculature, to name a few.
Despite the dominant role of retinal oxygen concentration in retinal vascular disease, no method exists for the diagnosis of oxygen concentration levels within the tissue of the retina in vivo. In addition, there are no methods for evaluating the benefits of treatment modalities to retinal oxygenation in retinal disease, such as panretinal photocoagulation. Moreover, because retinal tissue hypoxia is believed to precede changes in the retinal vasculature, it would be desirable to determine the retinal tissue oxygen concentration levels to allow early diagnosis of disease and treatment intervention prior to irreversible changes in the retinal tissue and vessels.
Because oxygen is generally transported to the retina and other various tissues in the body by the circulatory system, many tests or analyses for oxygen content in bodily tissues are conventionally directed to detecting the presence and/or viability of blood vessels in and around particular tissues in the body and blood flow within these vessels. Fluorescein angiography is one popularly used technique to determine the presence of blood vessels and blood flow in vivo as described, for example, in U.S. Pat. No. 3,893,447 of Hochheimer et al.; U.S. Pat. No. 4,249,825 of Shapiro; U.S. Pat. No. 4,304,720 of Dean et al.; and U.S. Pat. No. 4,341,223 of Lutz. However, these methods and apparatus are limited to the analysis of the blood vessels which is, at best, only an indirect approximation of the oxygen concentration levels in surrounding tissue.
Fluorescein angiography and similar fluorescence techniques are based on the knowledge that certain dyes are known to have the ability to fluoresce. The fluorescence of such dyes may be reduced by the addition reaction of certain substances, commonly called quenchers, to or with the dye. Fluorescent dyes having a known sensitivity to a particular quencher may be used as an indicator for the concentration of that particular quencher. When illuminated by a source beam of excitation light of a predetermined wavelength, the indicating dye typically emits a fluorescent beam of a wavelength different from the source beam and whose intensity is inversely proportional to the concentration of the particular quencher.
The relation between the concentration of the quencher and the reduction in fluorescence intensity is known generally as the Stern-Volmer relation which may be expressed as follows: EQU F.sub.o /F=1+k[Q]
where F.sub.o is the fluorescence intensity of a fluorescent indicator in the absence of a quencher, F is the fluorescence intensity of a fluorescent indicator in the presence of a quencher, k is the quenching constant specific to each pair of quencher/fluorescence indicator and [Q] is the concentration or partial pressure of the quencher.
Several fluorescent dyes sensitive to oxygen quenching are known, and their uses have been described by U.S. Pat. No. 4,041,932 of Fostick; U.S. Pat. No. 4,476,870 of Peterson et al.; U.S. Pat. No. 4,580,059 of Wolfbeis et al.; and U.S. Pat. No. 4,861,727 of Hauenstein et al.; and W. M. Vaughan et al. "Oxygen Quenching of Pyrenebutyric Acid Fluorescence in Water. A Dynamic Probe of the Microenvironment," Biochemistry, 9(3):464-73 (1970), for example. These methods of measuring oxygen concentration using fluorescent indicators are, however, limited to in vitro measurements of oxygen concentration levels or in vivo application for fluids only. Moreover, those in vivo methods for measuring fluids, principally blood, require the use of implantable or insertable sampling chambers through which the fluid or its constituents may be viewed or collected for analysis. No fluorescent indicator is present in the body fluid but, rather, is contained in the sampling chamber. In addition, these in vivo methods provide no direct measurement of oxygen concentration levels in bodily tissues.
In the view of the deficiencies of the prior art, it would be desirable to have a method for direct in vivo measurement of oxygen concentration levels in bodily fluids or tissues.